Method for determining the catalytic activity of factor IXa

ABSTRACT

A method for the determination of factor IXa in a sample solution using a measurable factor IXa substrate and a water-miscible alcohol and measuring the cleavage of the factor IXa substrate as a measure for factor IXa activity is suitable for the direct determination of factor IXa.

This application is the US national phase of PCT/EP97/05660, filed Oct.15, 1997.

The invention concerns a method for determining the catalytic activityof factor IXa. The method according to the invention is suitable forfinding factor IXa inhibitors (screening), for modulating bloodcoagulation (therapeutic application) and for determining factor IX andfactor IXa in body fluids (diagnostic application).

Blood plasma proteases play a role in blood coagulation, wound closureby fibrin formation as well as in fibrinolysis i.e. clot lysis. After aninjury the "injury signal" is amplified by the sequential activation(specific proteolysis) of inactive proenzymes to form active enzymeswhich initiates blood coagulation and ensures a rapid wound closure.Blood coagulation can be initiated by two paths, the intrinsic path inwhich all protein components are present in the blood and the extrinsicpath in which a membrane protein, the so-called tissue factor plays acritical role.

The molecular mechanism of blood homeostasis (blood coagulation,fibrinolysis and the regulation of this equilibrium) and the componentsthat are involved in this are comprehensively described in severalreview articles (Furie, B. and Furie, B. C., Cell 53 (1988) 505-518;Davie, E. W. et al., Biochem. 30 (1991) 10363-10379; Bergmeyer, H. U.(ed.): Methods of Enzymatic Analysis, Vol. V, chapter 3, 3rd ed.,Academic Press, New York (1983)).

The factors of the blood coagulation cascade are very complex proteins.As a rule they can only be isolated in a complicated manner from thenatural raw material source, the blood plasma, in a limited amount, withvarying quality, homogeneity and purity (Van Dam-Mieras, M.C.E. et al.,In: Bergmeyer, H. U. (ed.), Methods of Enzymatic Analysis, Vol. V, 3rded., page 365-394, Academic Press, New York (1983)). They play animportant role in the regulation of blood homeostasis which is theequilibrium between blood coagulation, clot formation and dissolution.This well-regulated system can become unbalanced by genetic defects suchas haemophilia A (defective factor VIII) and haemophilia B (defectivefactor IX). Acute disorders can lead to cardiac infarction, embolism andstroke.

There is therefore a need for substances which can influence the systemof blood coagulation and fibrinolysis according to the medicalrequirements. For example from blood isolated or recombinantly producedfactor VIII or factor IX are used to treat haemophilia A and B. tPA(tissue type plasminogen activator) and streptokinase (bacterialplasminogen activator) are used for example for clot lysis e.g. aftercardiac infarction. In addition to complex proteins, substances such ashirudin (peptide composed of 65 amino acids, thrombin inhibitor),heparin (heteroglycan, cofactor of endogenous inhibitors) and vitamin Kantagonists (inhibitors of γ-carboxylation of Glu residues of the GlAdomain) are also used to inhibit blood coagulation. However, theavailable substances are often still very expensive (protein factors)and not ideal with regard to their medical application (side effects) sothat there is a need for medicaments which can be used to specificallymodulate blood coagulation and clot lysis.

The search for new modulators (activators, inhibitors) of bloodcoagulation, fibrinolysis and homeostasis can for example be carried outby screening substance libraries and subsequently improving anidentified lead structure by drug modelling. For this it is necessarythat i) a suitable test and ii) the key protein(s) [target(s)] areavailable in an adequate amount and quality for screening and forcrystal structure investigations (e.g. improvement of the lead structureby the specific prediction of changes based on the 3D structure of theprotein component and lead structure).

Factor IXa (FIXa) is an interesting target for an inhibitor screening inorder to find inhibitors to modulate blood coagulation. The knownclinical picture of haemophilia B (factor IXa defect) warrants theassumption that specific factor IXa inhibitors are superior to knownthrombin inhibitors with regard to the quite considerable pleiotropicside-effects.

Previously, screening for FIXa inhibitor activity has failed due to theavailability and extremely low catalytic activity of FIXa.

The isolation of the inactive serine protease FIX (zymogen) from bloodplasma and the subsequent activation by proteolysis is difficult,time-consuming, expensive and often does not yield the desired amountand quality. Thus the plasma concentration of the inactive proteasezymogen FIX is only 0.5 mg/l (Furie, B. and Furie B. C., Cell 53 (1988)505-518). Moreover the protease preparations isolated from the plasmaand activated in vitro are often very heterogeneous and unstable.

The inactive FIX zymogen can for example be activated/converted usingpurified FXIa (Van Dam-Mieras, M.C.E.; Muller, A. D.; van Dieijen, G.;Hemker, H. C.: Blood coagulation factors II, V, VII, VIII, IX, X and XI:Determination with synthetic substrates. In: Bergmeyer, H. U. (ed.):Methods of Enzymatic Analysis, Vol. V, Enzymes 3: Peptidases,Proteinases and Their Inhibitors, page 365-394, 3rd ed., Academic Press,New York (1983)).

Blood plasma proteases are complex glycoproteins that belong to theserine protease family. They are synthesized in the liver as inactiveproenzymes (zymogens), secreted into the blood and are activated whenrequired by specific proteolysis i.e. by cleavage of one or two peptidebonds. They are structurally very similar with regard to the arrangementof their protein domains and their composition (Furie, B. and Furie, B.C., Cell 53 (1988) 505-518).

According to Furie B. and Furie, B. C. the proteases of the factor IXfamily (factor VII, IX, X and protein C) are composed of

a propeptide,

a GLA domain,

an aromatic amino acid stack domain,

two EGF domains (EGF1 and EGF2),

a zymogen activation domain (activation peptide, AP) and

a catalytic protease domain (CD).

Furthermore the blood plasma proteases are post-translationally modifiedduring secretion:

11-12 disulfide bridges

N- and/or O-glycosylation (GLA domain and activation peptide)

Bharadwaj, D. et al., J. Biol. Chem. 270 (1995) 6537-6542

Medved, L. V. et al., J. Biol. Chem. 270 (1995) 13652-13659

cleavage of the propeptide

γ-carboxylation of Glu residues (GLA domain)

β-hydroxylation of an Asp residue (EGF domains)

cleavage of the zymogen region (partially)

After activation of the zymogens (zymogenic form of the protein) byspecific cleavage of one or two peptide bonds (cleavage of an activationpeptide), the enzymatically active proteases are composed of two chainswhich, in accordance with their molecular weight, are referred to as theheavy and light chain. In the factor IX protease family the two chainsare held together by an intermolecular disulfide bridge between the EGF2domain and the protease domain. The zymogen-enzyme transformation(activation) leads to conformation changes within the protease domain.This enables an essential salt bridge necessary for the proteaseactivity to form between the N-terminal amino acid of the proteasedomain and an Asp residue within the protease domain. The N-terminalregion is very critical for this subgroup of serine proteases and shouldnot be modified. Only then is it possible for the typical active site ofthe serine proteases to form with the catalytic triad composed of Ser,Asp and His [Blow, D. M.: Acc. Chem. Res. 9 (1976) 145-152; Polgar, L.:In: Mechanisms of protease action. Boca Raton, Fla., CRC Press, chapter3 (1989)].

Blood plasma proteases can be produced in a classical manner byisolating the inactive zymogens from the blood and subsequentlyactivating them or they can be produced recombinantly by expressing thecorresponding cDNA in a suitable mammalian cell line or in yeast.

The production of coagulation factors by expression/secretion of thezymogens or active proteases by means of eukaryotic host/vector systemsis described for FvII: Hagen, F. S. et al., EP 0 200 421; Pedersen, A.H. et al., Biochem. 28 (1989) 9391-9336; FIX: Lin, S.-W. et al., J.Biol. Chem. 265 (1990) 144-150; FX: Wolf, D. L. et al., J. Biol. Chem.266 (1991) 13726-13730, Protein C: Bang, N. U. et al., EP 0 191 606.

As a rule host cells are used which are able to post-translationallymodify the coagulation factors like the native enzyme during thesecretion process. The zymogen-enzyme transformation is then carried outsubsequently during the downstream processing e.g. by using an activatorfrom snake venom in the case of prothrombin or factor X (Sheehan, J. P.et al., J. Biol. Chem. 268 (1993) 3639-3645; Fujikawa, K. et al.Biochem. 11 (1972) 4892-4898).

For the purpose of zymogen-enzyme activation in vivo (already duringsecretion), the natural zymogen cleavage sites or the entire activationpeptide were substituted by protease cleavage sites (several adjacentbasic amino acids) which can be cleaved by specifically cleavingproteases that occur naturally in the secretion path of the host cellsuch as e.g. Kex2 (yeast) or PACE (mammnalian cell lines). (FX: Wolf, D.L. et al., J. Biol. Chem. 266 (1991) 13726-13730; Prothrombin: Holly, R.D. and Foster, D. C., WO 93/13208).

The production of variants of coagulation factors (FX: Rezaie, A. R: etal., J. Biol. Chem. 268 (1993) 8176-8180); FIX: Zhong, D. G. et al.,Proc. Natl. Acad. Sci. USA 91 (1994) 3574-3578), mutants (FX: Rezaie, A.R. et al., J. Biol. Chem. 269 (1994) 21495-21499; Thrombin: Yee, J. etal., J. Biol. Chem. 269 (1994) 17965-17970); FVII: Nicolaisen, E. M. etal., WO 88/10295) and chimeras e.g. composed as FIX and FX (Lin, S.-W.et al., J. Biol. Chem. 265 (1990) 144-150; Hertzberg, M. S. et al., JBiol. Chem. 267 (1992) 14759-14766) by means of eukaryotic host/vectorsystems is also known.

However, expression in eukaryotic mammalian cell lines istime-consuming, limited with regard to expression output and expensive.In addition undesired post-translational modifications can occur.

The production of blood plasma proteases by expression in prokaryotesand subsequent renaturation of the expression product is described byThogersen, H. C. et al. (WO 94/18227). According to this FX variants arerenatured by means of a cyclic renaturation process in which theinactive FX protein is immobilized in a chromatographic column by meansof a metal chelate complex (poly(His)-affinity handle). A fusion proteinis used for this composed of a truncated FX variant (EGF1, EGF2 andprotease domain), an additional FXa protease recognition sequence and anattachment aid at the C-terminus of the catalytic domain composed of 6histidine residues.

Surprisingly it was found that the catalytic activity of factor IXa withrespect to substrates can be stimulated by alcohols and hence a verysensitive factor IXa test can be constructed in a simple manner.

Consequently the invention concerns a method for determining factor IXain a sample solution characterized in that a determinable factor IXasubstrate and an alcohol that is homogeneously miscible with water andforms one phase is added to the sample solution and the cleavage of thefactor IXa substrate is determined as a measure for the factor IXaactivity. It surprisingly turned out that the catalytic factor IXaactivity can be increased by more than 20 times by alcohols. As a resultit is possible to directly determine the factor IXa activity in samplesolutions. Factor IX can for example be determined in sample solutions,preferably body fluids such as plasma after activating factor IX tofactor IXa by means of Russels viper venom or by the protease isolatedfrom the snake venom (RVV-X protease).

A further subject matter of the invention is the use of thedetermination method according to the invention for factor IXa to screenfor substances which modulate (inhibit or activate) factor IXa activity.Consequently the invention concerns a method for identifying a substancewhich modulates the activity of factor IXa characterized in that

a) a factor IXa substrate is cleaved by a polypeptide with factor IXaactivity at a defined concentration in the presence of an alcohol andthe rate of cleavage of the said substrate is determined as a measurefor the factor IXa activity,

b) the said activity is measured in the presence of a test substance,

c) the activity is compared with and without the test substance and

d) the difference of the activity is used as a measure for the activitymodulation by the test substance.

The inhibition of factor IXa by a test substance is preferably tested.

Suitable factor IXa substrates are for example factor IXa substratesthat are described in EP-B 0 034 122. Substrates of theR-Xxx-Gly-Arg-pNA type are especially suitable in which Xxx represents ahydrophobic D-amino acid and pNA represents a determinable leavinggroup. R is defined analogously to EP-B 0 034 122. Substrates of thegeneral formula I from EP-B 0 034 122 in which R³ =R⁴ =H are preferred.

Preferred substrates are tripeptides with a hydrophobic D-amino acid atthe N-terminus and preferably a cyclohexyl-substituted hydrophobicD-amino acid is used. Cyclohexyl-glycine and cyclohexylalanine areparticularly preferred.

Further preferred substrates are for example substrates which are alsocleaved by factor Xa such as e.g. Chromozym X (Boehringer Mannheim GmbH,Moc-D-Nle-Gly-Arg-pNA) or Pefachrom tPA (Pentapharm Ltd., Basel, MeSO₂-D-HHT-Gly-Arg-pNA). Commercial (preferably chromogenic or fluorogenic)substrates that can be determined optically are preferably used. Thelatter are chromogenic peptides/substrates with a cleavable residue thatcan be easily determined by optical means (the p-nitroaniline residue ispreferred). The method according to the invention is preferably carriedout in a buffer solution. All buffers that are effective in a pH rangeof 7-10 (preferably between pH 7.5-9.0) can be used as buffersubstances. Tris buffer, triethanolamine and Tris-imidazole buffer arepreferred.

The determination of FIXa activity and the screening assay arepreferably carried out at 20-40° C., particularly preferably at roomtemperature and the amount of the cleaved optically determinable groupis determined photometrically or fluorometrically. The absorbance at 405nm is preferably determined. The enzymatic activity and the kineticconstants are determined from the linear initial slope according to theMichaelis-Menten equation.

Factor IX or the ratio of factor IX to factor IXa can be determinedanalogously after activation of factor IX to factor IXa. In order todetermine factor IX in plasma, factor IX is firstly activated to factorIXa by Russels viper venom (preferably 0.2 mg/ml) or RVV-X protease(preferably 0.1 mg/ml) in the presence of CaCl₂ (10 mmol/l) at 20-40°C., preferably at 37° C. After the activation is completed (preferably15 min), ethylene glycol and the cleavable substrate are added atconcentrations of 20-40% and 0.2-1 mmol/l respectively.

It has proven to be advantageous for a screening test to add factor IXaat a concentration of 0.02-0.5 μmol/l, preferably 0.05 μmol/l and thecleavage substrate at a concentration of 0.2-1 mmol/l with a testsubstance concentration in the μmol/l range. Ethylene glycol, ethanol ormethanol is preferably used as the alcohol. The concentration of thealcohol is preferably in the range of 20-40%. Native human factor IXa orporcine or bovine factor IXa or recombinantly produced human factor IXacan be used as factor IXa. A truncated human recombinant factor IXa thatis described in EP 96 109 288.9 is particularly preferably used.

Enzymatically active recombinant factor IXa can be produced byexpression of a corresponding DNA in prokaryotes, renaturation of theexpression product and enzymatic cleavage if it is composed of a FIXaserine protease domain (catalytic domain) N-terminally linked to an EGFdomain (EGF1 and/or EGF2) and a zymogen activation domain.

A non-glycosylated, enzymatically active factor IXa composed of thefollowing domains is preferably used:

a) the catalytic FIXa protease domain, N-terminally linked with

b) a zymogen activation domain, N-terminally linked with

c) an EGF1 and/or EGF2 domain (preferably EGF2 or EGF1 and EGF2).

A spacer with up to 50 amino acids is preferably inserted between thezymogen activation domain and the EGF domain (or the EGF domains). Whenthe zymogenic one chain form according to the invention is cleaved inthe zymogen activation domain, an active protein is obtained in a twochain form. Both chains are linked by an intermolecular disulfide bridgein the two chain form. The proteins according to the invention arepreferably composed of the EGF2 domain, the activation peptide and thecatalytic domain of factor IX. Such factor IX muteins are described inthe European Patent Application No. 96 110 959.2.

Any plasma can be used in the determination method according to theinvention and citrate plasma is preferred.

The method can be carried out at neutral or weakly alkaline pH valuespreferably in the pH range between 7.5 and 9.0. Physiologicallyacceptable buffers that are effective in this range can be used as thebuffer. In addition common stabilizers and preservatives for coagulationtests such as bovine serum albumin, merthiolate and such like can alsobe added.

In addition the method according to the invention and the reagentaccording to the invention can also contain a surfactant, preferably anon-ionic surfactant such as Tween 80®. In this case the concentrationis preferably 0.01-1 vol. %.

A further subject matter of the invention is a reagent for thedetermination of factor IXa which contains an optically measurablesubstrate that can be cleaved by factor IXa, alcohol and buffer in arange between pH 7 and 10.

The following examples, publications, the sequence protocol and thefigures further elucidate the invention, the protective scope of whichresults from the patent claims. The described methods are to beunderstood as examples which also still describe the subject matter ofthe invention even after modifications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the influence of ethanol and ethylene glycol on theactivity of recombinant rFIXa (0.48 μmol/l) and native FIXa (0.14μmol/l). pH 7.4; substrate: MeSO₂ -D-HHT-Gly-Arg-pNA (1.02mmol/l)--figure for table 2.

FIG. 2a shows the influence of ethylene glycol on the activity of rFIXa.S1=MeSO₂ -D-HHT-Gly-Arg-pNA; S2=MOC-D-Nle-Gly-Arg-pNA; S3=MeSO₂-D-CHG-Gly-Arg-pNA; S4=MeSO₂ -D-CHA-Gly-Arg-pNA; S5=MeSO₂-D-CHA-Gly-Arg-AMC; rFIXa=0.48 μmol/l; pH=7.4--figure for table 3.

FIG. 2b shows the influence of ethanol on the activity of rFIXa.S1=MeSO₂ -D-HHT-Gly-Arg-pNA; S2=MOC-D-Nle-Gly-Arg-pNA; S3=MeSO₂-D-CHG-Gly-Arg-pNA; S4=MeSO₂ -D-CHA-Gly-Arg-pNA; S5=MeSO₂-D-CHA-Gly-Arg-AMC; rFIXa=0.48 μmol/l; pH=7.4--figure for table 3

FIG. 3a shows the influence of ethanol on the activity of P-kallikrein,FXIIa, FXIa, FIXa, FXa and thrombin (pH 7.4; 1.02 mmol/l MeSO₂-D-HHT-Gly-Arg-pNA)--figure for table 5.

FIG. 3b shows the influence of ethylene glycol on the activity ofP-kallikrein, FXIIa, FXIa, FIXa, FXa and thrombin (pH 7.4; 1.02 mmol/lMeSO₂ -D-HHT-Gly-Arg-pNA)--figure for table 6.

Methods

Recombinant DNA Technique

Standard methods were used to manipulate DNA as described in Sambrook,J. et al. (1989) In: Molecular cloning: A laboratory manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Protein Determination

The protein concentration of the FIX variants was determined bydetermining the optical density (OD) at 280 nm using the molarextinction coefficients calculated on the basis of the amino acidsequence.

Expression Vector

The vector for the expression of the FIX variants is based on theexpression vector pSAM-CORE for core-streptavidin. The preparation anddescription of the plasmid PSAM-CORE is described in WO 93/09144.

The core-streptavidin gene was replaced by the gene of the desiredvariant in the PSAM-CORE vector.

EXAMPLE 1

Cloning the Catalytic Domain of the FIX Gene (Plasmid: pFIX-CD)

The FIX cDNA from bp position 690 to 1403, coding for the FIX proteasedomain from amino acid position 181 to 415 (cDNA sequence and amino acidsequence numbering according the publication of McGraw, R. A. et al.(Proc. Natl. Acad. Sci. USA 82 (1985) 2847-2851) was amplified using thePCR primers N1 (SEQ ID NO:1) and N2 (SEQ ID NO:2).

                  NcoI                                                              N1: 5'-AAAAAACCATGGTTGTTGGTGGAGAAGATGCCAAACC-3'                                              MetValValGlyGlyGluAspAlaLys                                     -              HindIII                                                       N2: 5'-AAAAAAAAGCTTCATTAAGTGAGCTTTGTTTTTTCCTTAATC-3'                    

and a commercially available human liver cDNA gene bank (vector: LambdaZAP® II) from the Stratagene Company (La Jolla, Calif., U.S.A.) astemplate DNA. The PCR primers introduced a singular NcoI cleavage siteand an ATG start codon at the 5' end of the coding region and a singularHindIII cleavage site at the 3' end of the coding region.

The ca. 730 bp long PCR product was digested with the restrictionendonucleases NcoI and HindIII and the ca. 720 bp long NcoI/HindIII-FIXfragment was ligated into the ca. 2.55 kbp long NcoI/HindIII-pSAM-COREvector fragment (see WO 93/09144) after purification by agarose gelelectrophoresis. The desired plasmid pFIX-CD was identified byrestriction mapping and the FIX cDNA sequence isolated by PCR waschecked by DNA sequencing.

EXAMPLE 2

Construction of the FIX Protease Gene with EGF2 Domain, activationpeptide and catalytic domain (plasmid: pFIX-EGF2-AP-CD)

The FIX cDNA from bp position 402 to 986, coding for the EGF2 domain,the activation peptide and the N-terminal region of the FIXa proteasedomain from amino acid position 85 to 278 (McGraw et al. Proc. Natl.Acad. Sci. USA 82 (1985) 2847-2851) was amplified using the PCR primersN3 (SEQ ID NO:3) and N4 (SEQ ID NO:4).

                  NcoI                                                              N3: 5'-AAAAAACCATGGATGTAACATGTAACATTAAGAATGGCA-3'                                            MetAspValThrCysAsnIleLysAsnGly                                  - N4: 5'-GGGTTCGTCCAGTTCCAGAAGGGC-3'                                   

and a commercially available human liver CDNA gene bank (vector: LambdaZAP® II) from the Stratagene Company (La Jolla, Calif., U.S.A.) astemplate DNA. The PCR primer N3 introduced an ATG start codon and asingular NcoI cleavage site at the 5' end of the coding region.

The ca. 590 bp long PCR product was digested with the restrictionendonucleases NcoI and BsmI and the ca. 360 bp longNcoI/BsmI-FIX-EGF2-AP fragment was ligated into the ca. 3.2 kbp longNcoI/BsmI-pFIX-CD vector fragment (example 1) after purification byagarose gel electrophoresis. The desired plasmid pFIX-EGF2-AP-CD wasidentified by restriction mapping and the FIX cDNA sequence isolated byPCR was checked by DNA sequencing.

EXAMPLE 3

a) Expression of the FIXa Protease Gene in E. coli

In order to express the FIX gene containing the activation peptide, theE. coli K12 strain UT5600 (Grodberg, J. and Dunn, J. J. J. Bacteriol.170 (1988) 1245-1253) was transformed with the expression plasmidpFIX-EGF2-AP-CD (ampicillin resistance) described in example 2 and withthe lacI^(q) repressor plasmid pUBS520 (kanamycin resistance,preparation and description see: Brinkmann, U. et al., Gene 85 (1989)109-114). Other E. coli strains known and available to a person skilledin the art such as HB101 and E. coli B can also be used instead of thestrain UT5600.

The UT5600/pUBS520 cells transformed with the expression plasmidpFIX-EGF2-AP-CD were cultured in a shaking culture in DYT medium (1%(w/v) yeast extract, 1% (w/v) Bacto Tryptone, Difco and 0.5% NaCl)containing 50-100 mg/l ampicillin and 50 mg/l kanamycin at 37° C. up toan optical density at 550 nm (OD₅₅₀) of 0.6-0.9 and subsequently inducedwith IPTG (final concentration 1-5 mmol/l). After an induction phase of4-8 hours (h) at 37° C., the cells were harvested by centrifugation(Sorvall RC-5B centrifuge, GS3 rotor, 6000 rpm, 15 min), washed with 50mmol/l Tris-HCl buffer pH 7.2 and stored at -20° C. until furtherprocessing. The cell yield from a 1 l shaking culture was 4-5 g (wetweight).

b) Expression Analysis

The expression of the UT5600/pUBS520 cells transformed with the plasmidpFIX-EGF2-AP-CD was analysed. For this purpose cell pellets from in eachcase 1 ml centrifuged culture medium were resuspended in 0.25 ml 10mmol/l Tris-HCl, pH 7.2 and the cells were lysed by ultrasonic treatment(2 pulses of 30 s at 50% intensity) using a Sonifier® Cell Disruptor B15from the Branson Company (Heusenstamm, Germany). The insoluble cellcomponents were sedimented (Eppendorf 5415 centrifuge, 14000 rpm, 5 min)and 1/5 volumes (vol) 5×SDS sample buffer (1×SDS sample buffer: 50mmol/l Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol,0.001% bromophenol blue) was added to the supernatant. The insolublecell debris fraction (pellet) was resuspended in 0.3 ml 1×SDS samplebuffer containing 6-8 M urea, the samples were incubated for 5 min at95° C. and centrifuged again. Afterwards the proteins were separated bySDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, U.K., Nature 227(1970) 680-685) and stained with Coomassie Brilliant Blue R dye.

The FIX variant synthesized in E. coli was homogeneous and wasexclusively found in the insoluble cell debris fraction (inclusionbodies, IBs). The expression yield was 10-50% relative to the total E.coli protein.

EXAMPLE 4

Cell Lysis, Solubilization and Renaturation

a) Cell Lysis and Preparation of Inclusion Bodies (IBs)

The cell pellet from 3 l shaking culture (ca. 15 g wet weight) wasresuspended in 75 ml 50 mmol/l Tris-HCl, pH 7.2. The suspension wasadmixed with 0.25 mg/ml lysozyme and it was incubated for 30 min at 0°C. After addition of 2 mmol/l MgCl₂ and 10 μg/ml DNase I (BoehringerMannhein GmbH, catalogue No. 104159) the cells were disruptedmechanically by means of high pressure dispersion in a French® Pressfrom the SLM Amico Company (Urbana, Ill., USA). Subsequently the DNA wasdigested for 30 min at room temperature (RT). 37.5 ml 50 mmol/l Tris-HClpH 7.2, 60 mmol/l EDTA, 1.5 mol/l NaCl, 6% Triton X-100 was added to thepreparation, it was incubated for a further 30 min at RT and centrifugedin a Sorvall RC-5B centrifuge (GSA Rotor, 12000 rpm, 15 min). Thesupernatant was discarded, 100 ml 50 mmol/l Tris-HCl, pH 7.2, 20 mmol/lEDTA was added to the pellet, it was incubated for 30 min while stirringat 4° C. and again sedimented. The last wash step was repeated. Thepurified IBs (1.5-2.0 g wet weight, 25-30% dry mass, 100-150 mgprotease) were stored at -20° C. until further processing.

b) Solubilization and Derivatization of the IBs

The purified IBs were suspended within 1 to 3 hours at room temperaturewhile stirring at a concentration of 100 mg IB pellet (wet weight)/mlcorresponding to 5-10 mg/ml protein in 6 mol/l guanidinium-HCl, 100mmol/l Tris-HCl, 20 mmol/l EDTA, 150 mmol/l GSSG and 15 mmol/l GSH, pH8.0. Afterwards the pH was adjusted to pH 5.0 and the insolublecomponents were separated by centrifugation (Sorvall RC-5B centrifuge,SS34 rotor, 16000 rpm, 10 min). The supernatant was dialysed for 24hours at 4° C. against 100 vol. 4-6 mol/l guanidinium-HCl pH 5.0.

c) Renaturation

The renaturation of the proteins solubilized in 6 mol/l guanidinium-HCland derivatized with GSSG/GSH was carried out at 4° C. by repeated (e.g.3-fold) addition of 0.5 ml IB solubilisate/derivative in each case to 50ml 50 mmol/l Tris-HCl, 0.5 mol/l arginine, 20 mmol/l CaCl₂, 1 mmol/lEDTA and 0.5 mmol/l cysteine (pH 8.5) at intervals of 24 hours andsubsequent incubation for 48 hours at 4° C. After completion of therenaturation reaction the insoluble components were separated byfiltration with a filtration apparatus from the Satorius Company(Gottingen, Germany) equipped with a deep bed filter K 250 from theSeitz Company (Bad Kreuznach, Germany).

d) Concentration and Dialysis of the Renaturation Preparations

The clear supernatant containing protease was concentrated 10-15-fold bycross-flow filtration in a Minisette (membrane type: Omega 10K) from theFiltron Company (Karlstein, Germany) and dialysed for 24 hours at 4° C.against 100 volumes 20 mmol/l Tris-HCl and 50 mmol/l NaCl, pH 7.2 toremove guanidinium-HCl and arginine. Precipitated protein was removed bycentrifugation (Sorvall RC-5B centrifuge, SS34 rotor, 16000 rpm, 20 min)and the clear supernatant was filtered with a Nalgene® disposablefiltration unit (pore diameter: 0.2 μm) from the Nalge Company(Rochester, N.Y., USA).

EXAMPLE 5

Purification of the Renatured FIX Variant

The FIX variant from the renaturation preparation can, if required, befurther purified with chromatographic methods which are known to aperson skilled in the art.

a) Purification of the FIX Variant by Ion Exchange Chromatography onQ-Sepharose-ff

The concentrated renaturation preparation that had been dialysed against20 mmol/l Tris-HCl and 50 mmol/l NaCl, pH 8.0 was applied to aQ-Sepharose ff column (1.5×11 cm, V=20 ml; loading capacity: 10 mgprotein/ml gel) from the Pharmacia Biotech Company (Freiburg, Germany)(2 column volumes/hour, 2 CV/h) equilibrated with the same buffer and itwas washed with the equilibration buffer until the absorbance of theeluate at 280 nm had reached the blank value of the buffer. The boundmaterial was eluted by a gradient of 50-500 mmol/l NaCl in 20 mmol/lTris-HCl, pH 8.0 (2 CV/ h). The FIX variant was eluted at an NaClconcentration of 100-150 mmol/l. The fractions containing FIX wereidentified by non-reducing and reducing SDS PAGE and the elution peakwas pooled.

b) Final Purification of the FIX Variant by Ion Exchange Chromatographyon Heparin-Sepharose CL-6B

After chromatography on Q-Sepharose ff, the combined fractionscontaining FIX were directly applied (2 CV/h) to a heparin-SepharoseCL-6B column (1.5×11 cm, V=20 ml, loading capacity: 1 mg protein/ml gel)from the Pharmacia Biotech Company (Freiburg, Germany) that had beenequilibrated with 20 mmol/l Tris-HCl and 200 mmol/l NaCl, pH 8.0.Afterwards it was washed with equilibration buffer until the absorbanceof the eluate at 280 nm reached the blank value for the buffer. Thebound material was eluted by a gradient of 0.2-1.0 mol/l NaCl in 20mmol/l Tris-HCl, pH 8.0 (2 CV/h). The FIX variants were eluted at anNaCl concentration of 500-600 mmol/l. The fractions containing FIX wereidentified by non-reducing and reducing SDS PAGE, the elution peak wascombined and dialysed against 20 mmol/l Tris-HCl, 50-200 mmol/l NaCl, 5mmol/l CaCl₂, pH 7.8.

EXAMPLE 6

Activation and Purification of the FIX Variant

The renatured purified FIX variant was activated with purified Russellsviper venom (RVV-X) protease. The RVV-X protease was, as described inthe publication by Esmon, C. T. (prothrombin activation, doctoraldissertation, Washington University, St. Louis, Mo. (1973)), purifiedfrom the commercially available snake venom lyophilisate from SigmaAldrich Chemie GmbH Co. (Deisenhofen, Germany) by gel filtrationfollowed by ion exchange chromatography on Q-Sepharose ff .

a) Activation and Purification of the Renatured FIX Variant

The FIX variant was digested at 25° C. at a concentration of 0.5 to 2.0mg/ml and a protease/substrate ratio of 1:10 to 1:20 in 20 mmol/1Tris-HCl, 50 mmol/l NaCl, 10 mmol/l CaCl₂, pH 7.8. The time course ofthe enzymatic FIX activation was monitored by determining the activitywith a chromogenic substrate (see example 13a) until the digestion wascompleted (plateau, maximum activation). For this purpose samples (10 to100 μl) were taken from the reaction mixture at intervals of 3-4 h overa period of up to 24 hours and the generated FIXa activity wasdetermined. After reaching the activation plateau, the activationpreparation was purified by negative chromatography on Q-Sepharose-ff.

RVV-X and the non-activated FIX variant bind under the given conditionsto Q-Sepharose-ff, but the activated FIXa variant does not.

The activation preparation was applied (2 CV/h) to a Q-Sepharose-ffcolumn (1.0×10 cm, V=8 ml) from the Pharmacia Biotech Company (Freiburg,Germany) which had been equilibrated with 20 mmol/l Tris-HCl, 50 mmol/lNaCl, pH 7.8 and the column was developed with equilibration bufferwhile fractionating. The fractions containing protease were identifiedby non-reducing and reducing SDS PAGE and activity determination.

EXAMPLE 7

Characterization of the Purified Protease Variants

a) Activity Test

The activity of the FIXa variants was determined using the chromogenicsubstrate Chromozym X (Boehringer Mannheim GmbH, Mannheim, Germany, cat.No. 789763). 10-100 μl sample was made up to 200 μl with 190-100 μl 50mmol/l Tris-HCl, 150 mmol/l NaCl, 5 mmol/l CaCl₂, 0.1% PEG 8000, pH 8.0,admixed with 20 μl Chromozym X (0.5-40 mmol) and measured at awavelength of 405 nm and RT against a reagent blank value in an ELISAreader. The activity and the kinetic constants were determined from thelinear initial slope according to the Michaelis Menten equation.

b) SDS PAGE

Oligomer and aggregate formation by intermolecular disulfide bridgeformation as well as the homogeneity and purity of the renaturedactivated and purified FIXa protease variant were examined bynon-reducing (minus mercaptoethanol) and reducing (plus mercaptoethanol)SDS PAGE (Laemmli, UK, Nature 227 (1970) 680-685).

EXAMPLE 8

Factor IXa Test

The FIXa test was carried out with recombinantly produced native humanFIXa as a sample and the peptide substrate MeSO₂ -D-HHT-Gly-Arg-pNA(Pefachrom tPA, Pentapharm Ltd., Basel, Switzerland) or otherchromogenic/fluorogenic substrates of the R-D-Xxx-Gly-Arg-pNA type (EP-B0 034 122) in Tris-HCl buffer and the substances to be tested (alcohol,solvents and inhibitors). The reaction was started by adding FIXa.

Test Principle

FIXa

MeSO₂ -D-HHT-Gly-Arg-pNA→MeSO₂ -D-HHT-Gly-Arg+pNA measurement signal:pNA

FIXa substrates: MOC-D-Nle-Gly-Arg-pNA (Chromozym X)

MeSO₂ -D-HHT -Gly-Arg -pNA (Pefachrom tPA)

MeSO₂ -D-CHG-Gly-Arg-pNA

MeSO₂ -D-CHA-Gly-Arg-pNA

MeSO₂ -D-CHA-Gly-Arg-AMC

Abbreviations: pNA, p-nitroaniline

AMC, 7-amino-4-methyl-coumaryl

MOC, methyloxycarbonyl

HHT, hexahydrotyrosine

CHG, cyclohexylglycine

CHA, cyclohexylalanine

General Test Mixture

200 μl buffer

50-100 mmol/l Tris-HCl, pH 7-9.5

100-200 mmol/l NaCl

5 mmol/l CaCl₂

with additives (alcohols, solvents and inhibitors)

+25 μl peptide substrate (1-20 mmol/l)

+20 μl FIXa (0.02-0.5 4 μmol/l)

The test mixture was incubated at room temperature (25° C.) in amicrotitre plate and the change in absorbance (ΔA/min) was measured at405 nm using an ELISA reader. The activity and the kinetic constantswere determined from the linear initial slope according to theMichaelis-Menten equation.

EXAMPLE 9

Influence of Alcohols and Organic Solvents on the Activity of rFIXa

The influence of various alcohols that can be completely mixed withwater to form a single phase (methanol, ethanol, n-propanol, i-propanol,t-butanol, ethylene glycol and glycerol) and organic solvents[acetonitrile, DMSO (dimethylsulfoxide) and DMF (dimethylformamide) andreadily soluble multivalent alcohols (m-erythritol, PEG (polyethyleneglycol)] on the activity of rFIXa was examined in relation to theconcentration of the alcohols or organic solvents (0-50%).

Test Mixture (Concentration in the Test)

41 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/ CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.48 μmol/l rFIXa (human)

0-50% (alcohol or organic solvent)

The results are shown in table 1. The ratio V/V₀ was calculated toillustrate the stimulation of the catalytic activity of rFIXa.

V₀, reaction rate without additives

V, reaction rate in the presence of additives

                  TABLE 1                                                         ______________________________________                                               V/V.sub.0                                                              Additive [%]                                                                           0      8      16   25    33    41    50                              ______________________________________                                        Methanol 1      2.16   3.46 3.71  2.83  1.55  0.588                             Ethanol 1 2.99 5.52 5.57 3.35 1.48 0.606                                      n-Propanol 1 3.43 2.26 0.339 0.186 0.056 0.056                                i-Propanol 1 2.17 2.48 1.18 0.346 0.164 0.164                                 t-Butanol 1 2.05 1.67 0.702 0.368 0.263 0.263                                 Ethylene glycol 1 2.91 6.05 10.27 12.68 9.68 3.41                             Glycerol 1 1.12 1.64 2.47 3.38 4.82 5.91                                      m-Erythritol* 1 1.16 1.35 1.49 1.56 1.64 1.77                                 PEG* 1 0.691 0.410 0.352 0.309 0.216 0.173                                    Acetonitrile 1 0.828 0.314 0 0 0 0                                            DMSO 1 1.27 1.45 1.59 1.43 0.922 0.157                                        DMF 1 0.649 0.514 0 0 0 0                                                   ______________________________________                                         *g/100 ml                                                                

Result

The activity of rFIXa is stimulated ca. 5-15-fold by some alcohols(ethylene glycol>ethanol>methanol) in the concentration range of 15-40%.Glycerol does not stimulate until higher concentrations (25-50%) whereasm-erythritol only has a slight effect. In contrast polyethylene glycol(PEG) inhibits the activity of rFIXa. Of the solvents which contain noOH group only dimethylsulfoxide (DMSO) does not inhibit rFIXa up to 30%whereas dimethylformamide (DMF) and acetonitrile inactivate the enzyme.

EXAMPLE 10

Influence of Ethanol and Ethylene Glycol on the Activity of RecombinantHuman FIXa and Native FIXa

It was examined whether the observed stimulation of rFIXa (active rFXavariant composed of an EGFII domain, processed activation peptide andcatalytic domain) by ethanol and ethylene glycol also occurs with nativeFIXa (human).

Test Mixture (Concentration in the Test)

41 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.48 μmol/l human rFIXa or

0.14 μmol/l native FIXa (human)

0-50% ethanol or ethylene glycol

The results are summarized in table 2 and shown in FIG. 1.

                  TABLE 2                                                         ______________________________________                                               V/V.sub.0                                                              Additive [%]                                                                           0      8      16   25    33    41    50                              ______________________________________                                        Human rFIXa                                                                     ethanol 1 2.99 5.52 5.57 3.35 1.48 0.61                                       ethylene glycol 1 2.91 6.05 10.27 12.68 9.68 3.41                             Native FIXa                                                                   (human)                                                                       ethanol 1 3.40 5.46 5.96 4.82 1.69 1.17                                       ethylene glycol 1 3.13 6.19 9.36 12.21 8.29 3.92                            ______________________________________                                    

Result

No differences were found for recombinant human rFIXa and native humanFIXa with regard to activation by ethanol and ethylene glycol.

EXAMPLE 11

Influence of Ethanol and Ethylene Glycol on the Activity of rFIXaTowards Chromogenic and Fluorogenic Peptide Substrates

The stimulation of human rFIXa by ethanol and ethylene glycol wasexamined using the peptide substrates MeSO₂ -D-HHT-Gly-Arg-pNA(Pefachrom tPA, Pentapharm Ltd., Basel, Switzerland),MOC-D-Nle-Gly-Arg-pNA (Chromozym X, Boehringer Mannheim GmbH, Mannheim,Germany, Cat.No. 789763), MeSO₂ -D-CHG-Gly-Arg-pNA (Pentapharm Ltd.,Basel, Switzerland), MeSO₂ -D-CHA-Gly-Arg-pNA (Pentapharm Ltd., Basel,Switzerland) and MeSO₂ -D-CHA-Gly-Arg-AMC (Pentapharm Ltd., Basel,Switzerland).

Test Mixture Chromogenic (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

0-50% ethylene glycol

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA,

0.48 μmol/l rFIXa (human)

or

0.82 mmol/l MOC-D-Nle-Gly-Arg-pNA

0.48 μmol/l rFIXa (human)

or

1.02 mmol/l MeSO₂ -D-CHG-Gly-Arg-pNA

0.28 μmol/l rFIXa (human)

or

1.02 mmol/l MeSO₂ -D-CHA-Gly-Arg-pNA

0.48 μmol/l rFIXa (human)

Test Mixture Fluorogenic (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

0-50% ethylene glycol

0.51 mmol/l MeSO₂ -D-CHA-Gly-Arg-AMC

0.08 μmol/l rFIXa (human)

The results are summarized in table 3, the results with ethylene glycolare shown in FIG. 2a and the results with ethanol are shown in FIG. 2b.

                  TABLE 3                                                         ______________________________________                                                 V/V.sub.0                                                              Substrate Additive [%]                                                      Additive 0      8      16   25    33    41    50                              ______________________________________                                        MeSO.sub.2 --D--                                                                HHT--Gly--                                                                    Arg--pNA                                                                      ethanol 1 2.99 5.52 5.57 3.35 1.48 0.60                                       ethylene glycol 1 2.91 6.05 10.27 12.68 9.68 3.41                             MOC--D--                                                                      Nle--Gly--                                                                    Arg--pNA                                                                      ethanol 1 3.63 7.28 9.95 7.51 3.15 1.39                                       ethylene glycol 1 3.91 8.85 17.07 22.42 17.80 6.96                            MeSO.sub.2 --D--                                                              CHG--Gly--                                                                    Arg--pNA                                                                      ethanol 1 3.33 6.02 6.16 4.50 1.74 0.87                                       ethylene glycol 1 5.47 13.13 19.37 23.20 17.40 5.67                           MeSO.sub.2 --D--                                                              CHA--Gly--                                                                    Arg--pNA                                                                      ethanol 1 3.30 5.40 5.38 4.30 1.48 0.89                                       ethylene glycol 1 2.84 6.26 9.34 11.18 7.54 2.56                              MeSO.sub.2 --D--                                                              CHA--Gly--                                                                    Arg--AMC                                                                      ethanol 1 2.09 2.50 1.37 1.01 0.70 0.58                                       ethylene glycol 1 2.55 5.59 8.40 8.90 5.79 1.44                             ______________________________________                                    

Result

The stimulation of rFIXa by ethanol and ethylene glycol expressed as thequotient V/V₀ is further increased when using the peptide substratesMOC-D-Nle-Gly-Arg-pNA and MeSO₂ -D-CHG-Gly-Arg-pNA instead of MeSO₂-D-HHT-Gly-Arg-pNA. Above 25% ethylene glycol the quotient V/V₀ is1.5-2-fold higher than with the substrate MeSO₂ -D-HHT-Gly-Arg-pNA.

The cleavage of the fluorogenic substrate MeSO₂ -D-CHA-Gly-Arg-AMC isalso stimulated by alcohols in a comparable manner. The cleavage oftripeptides with the sequence MeSO₂ -D-CHA-Gly-Arg and C-terminal AMC orpNA is increased by 9 and 11 times respectively by ethylene glycol(33%). Due to the higher sensitivity, much less (1/10) rFIXa must beused to cleave the fluorogenic substrate MeSO₂ -D-CHA-Gly-Arg-AMC.

EXAMPLE 12

Influence of Ethylene Glycol on the Kinetics of Cleavage of theChromogenic Peptide Substrates MeSO₂ -D-HHT-Gly-Arg-pNA,MOC-D-Nle-Gly-Arg-pNA and MeSO₂ -D-CHG-Gly-Arg-pNA by Recombinant HumanFIXa and Native FIXa

Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

0-50% ethylene glycol

MeSO₂ -D-HHT-Gly-Arg-pNA (0.102-1.02 mmol/l)

0.48 μmol/l rFIXa (human)

or

MeSO₂ -D-HHT-Gly-Arg-pNA (0.102-1.02 mmol/l)

0.29 μmol/l native FIXa (human)

or

MOC-D-Nle-Gly-Arg-pNA (0.102-1.02 mmol/l)

0.48 μmol/l rFIXa (human)

or

MeSO₂ -D-CHG-Gly-Arg-pNA (0.102-1.02 mmol/l)

0.28 μmol/l rFIXa (human)

The kinetic constants (K_(cat) /K_(m)) were determined graphicallyaccording to Lineweaver Burk (K_(m) and K_(cat) cannot be determinedseparately since the straight lines go through the origin).

                  TABLE 4a                                                        ______________________________________                                        Substrate: MeSO.sub.2 --D--HHT--Gly--Arg--pNA                                          k.sub.kat /K.sub.m [l/mmol*s]                                          ethylene glycol concentration [%]                                           Enzyme   0      8      16   25    33    41    50                              ______________________________________                                        rFIXa    n.l.   0.348  0.515                                                                              1.018 1.018 0.692 0.172                             native FIXa n.l 0.299 0.737 1.169 1.395 0.922 0.390                         ______________________________________                                    

                  TABLE 4b                                                        ______________________________________                                        Different substrates, rFIXa                                                               k.sub.kat /K.sub.m [l/mmol*s]                                       ethylene glycol concentration [%]                                           Substrate   0      8      16   25   33   41   50                              ______________________________________                                        MeSO.sub.2 --D--HHT--                                                                     n.l.   0.348  0.515                                                                              1.018                                                                              1.018                                                                              0.692                                                                              0.172                             Gly--Arg--pNA                                                                 MOC--D--Nle-- n.l. n.l. 0.326 0.565 0.552 0.360 0.148                         Gly--Arg--pNA                                                                 MeSO.sub.2 --D--CHG-- n.l. 0.637 1.974 2.880 3.480 1.929 0.576                Gly--Arg--pNA                                                               ______________________________________                                         n.l. = nollinear                                                         

Result

The stimulation of the cleavage of chromogenic peptide substratescatalysed by recombinant or native human FIXa is also found for thekinetic constants, K_(cat) /K_(m) increases up to an ethylene glycolconcentration of 33%. In the case of the substrate MeSO₂-D-CHG-Gly-Arg-pNA the highest catalytic activity i.e. the highest valuefor K_(cat) /K_(m) is found at 33% ethylene glycol. In the case of theuninfluenced reaction the plot is not linear and no constant can bedetermined.

EXAMPLE 13

Influence of Ethanol and Ethylene Glycol on the Activity of FIXa inComparison with Plasma Kallikrein, FXIIa, FXIa, FXa and Thrombin

FIXa Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.14 μmol/l native FIXa (human)

0-50% ethanol or ethylene glycol

Plasma Kallikrein Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.0176 U/ml plasma kallikrein (human)

0-50% ethanol or ethylene glycol

FXIIa Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.0045 U/ml FXIIa (human)

0-50% ethanol or ethylene glycol

FXIa Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.0012 μmol/l FXIa (human)

0-50% ethanol or ethylene glycol

FXa Test Mixture (concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.088 μmol/l FXa (bovine)

0-50% ethanol or ethylene glycol

Thrombin Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA

0.045 μmol/l thrombin (bovine)

0-50% ethanol or ethylene glycol

The results are summarized in tables 5 and 6, the effect of ethanol isshown in FIG. 3a and the effect of ethylene glycol is shown in FIG. 3b.

                  TABLE 5                                                         ______________________________________                                               V/V.sub.0                                                                ethanol concentration [%]                                                   Protease 0      8      16   25    33    41    50                              ______________________________________                                        PKallikrein                                                                            1      1.48   1.10 0.377 0.280 0.215 0.174                             FXIIa 1 0.904 0.579 0.187 0.051 0.009 0                                       FXIa 1 1.08 0.845 0.657 0.469 0.353 0.258                                     FIXa 1 3.40 5.46 5.96 4.82 1.69 1.17                                          FXa 1 0.899 0.752 0.510 0.377 0.203 0.086                                     thrombin 1 0.846 0.685 0.507 0.343 0.184 0.087                              ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                               V/V.sub.0                                                                ethylene glycol concentration [%]                                           Protease 0      8      16   25    33    41    50                              ______________________________________                                        PKallikrein                                                                            1      1.05   0.763                                                                              0.676 0.536 0.478 0.290                             F XIIa 1 0.892 0.729 0.551 0.355 0.187 0.103                                  FXIa 1 0.922 0.790 0.569 0.422 0.275 0.165                                    FIXa 1 3.13 6.19 9.36 12.21 8.29 3.92                                         FXa 1 0.973 0.902 0.741 0.558 0.347 0.191                                     thrombin 1 0.924 0.862 0.763 0.653 0.508 0.395                              ______________________________________                                    

Result

Among the coagulation factors of the intrinsic activation path--plasmakallikrein, FXIIa, FXIa, FIXa, FXa and thrombin--only FIXa is activatedby ethanol and ethylene glycol. A slight activation is observed in thepresence of 10-20% ethanol in the case of plasma kallikrein.

EXAMPLE 14

Influence of the pH on the Activity of rFIXa and Native FIXa in thePresence of Ethylene Glycol

Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.0-10.0

82 mmol/l NaCl

4.1 mmol/l CaCl₂

1.02 mmol/l MeSO₂ -D-HHT-Gly-Arg-pNA, MOC-D-Nle-Gly-Arg-pNA or MeSO₂-D-CHG-Gly-Arg-pNA

25% ethylene glycol

0.28 μmol/l rFIXa (human)

or

0.14 μmol/l native FIXa (human)

The results are shown in Table 7a-d.

                  TABLE 7a                                                        ______________________________________                                        Substrate: MeSO.sub.2 --D--HHT--Gly--Arg-pNA, rFIXa (human)                     Addi-                                                                         tive/ mA/min                                                                pH    7.0    7.5    8.0  8.25 8.5  8.75 9.0  9.5  10.0                        ______________________________________                                        without                                                                             6.5    10.7   16.0 17.0 16.3 16.3 16.0 12.9 10.4                          ethylene 56.5 124.5 173.6 176.0 182.1 170.7 164.1 112.0 110.1                 glycol                                                                        [25%]                                                                       ______________________________________                                    

                  TABLE 7b                                                        ______________________________________                                        Substrate: MeSO.sub.2 --D--HHT--Gly--Arg--pNA, native FIXa (human)              Addi-                                                                                  tive/ mA/min                                                       pH    7.0    7.5    8.0  8.25 8.5  8.75 9.0  9.5  10.0                        ______________________________________                                        without                                                                             3.3    6.8    10.0 10.5 10.3 9.9  9.3  7.4  6.2                           ethylene 23.7 63.0 101.0 106.7 115.4 110.9 105.6 100.6 77.2                   glycol                                                                        [25%]                                                                       ______________________________________                                    

                  TABLE 7c                                                        ______________________________________                                        Substrate: MOC--D--Nle--Gly--Arg--pNA, rFIXa (human)                            Addi-                                                                         tive/ mA/min                                                                pH    7.0    7.5    8.0  8.25 8.5  8.75 9.0  9.5  10.0                        ______________________________________                                        without                                                                             3.1    5.2    6.8  7.3  7.9  7.1  7.1  7.0  5.2                           ethylene 31.5 70.1 99.7 102.7 104.3 101.6 97.3 70.7 68.3                      glycol                                                                        [25%]                                                                       ______________________________________                                    

                  TABLE 7d                                                        ______________________________________                                        Substrate: MeSO.sub.2 --D--CHG--Gly--Arg--pNA, rFIXa (human)                    Addi-                                                                         tive/ mA/min                                                                pH    7.0    7.5    8.0  8.25 8.5  8.75 9.0  9.5  10.0                        ______________________________________                                        without                                                                             6.5    11.3   18.1 18.8 19.6 20.3 18.4 14.8 11.3                          ethylene 154.7 289.8 372.4 382.2 389.8 395.6 373.3 255.6 244.5                glycol                                                                        [25%]                                                                       ______________________________________                                    

Result

The pH dependency of the catalytic activity of rFIXa is not altered byalcohols as demonstrated with ethylene glycol as an example. Thus thecleavage rates increased in the same ratio between pH 7.0 and 10.0 inthe presence of 25% ethylene glycol. The cleavage rates in the presenceof the substrate MeSO₂ -D-HHT-Gly-Arg-pNA increased 10-fold, in the caseof MOC-D-Nle-Gly-Arg-pNA and MeSO₂ -D-CHG-Gly-Arg-pNA the increases were14-fold and 20-fold respectively. The optimal pH range is between pH8.25 and 8.75. The substrate MeSO₂ -D-CHG-Gly-Arg-pNA is cleaved mosteffectively.

EXAMPLE 15

Screening Test for FIXa Inhibitors

Specific FIXa inhibitors can be identified by the inhibition of FIXaactivity. For this purpose the FIXa activity is determined with asubstrate of the R-D-Xxx-Gly-Arg-pNA type (Xxx=hydrophobic amino acid)in the absence and presence of the substance to be tested or of asubstance mixture and the percentage inhibition is calculated from thequotients. The inhibition constant K_(i) is determined from theinhibition kinetics. The measurement signal is amplified by the presenceof high concentrations of certain alcohols (preferably ethylene glycol).

Test Principle

FIXa

R-D-Xxx-Gly-Arg-pNA→R-D-Xxx-Gly-Arg+pNA measurement signal: pNA

FIXa substrates: MeSO₂ -D-HHT-Gly-Arg-pNA (Pefachrom tPA)MOC-D-Nle-Gly-Arg-pNA (Chromozym X) MeSO₂ -D-CHG-Gly-Arg-pNA

FIXa: recombinantly produced rFIXa (human)

Test mixture

200 μl buffer (100 mmol/l Tris-HCl, 100 mmol/l NaCl, 5 mmol/l CaCl₂,20-40% alcohol, pH 7-9) the buffer contains the inhibitor at variousconcentrations

+25 μl peptide substrate

+20 μl rFIXa (human)

The test mixture was incubated at RT in a microtitre plate and thechange in absorbance (ΔA/min) at 405 nm was determined with an ELISAreader. The reaction can also be terminated after 5-10 min by stoppingwith acetic acid (25 μl, 50%), the absorbance is determined at 405 nmagainst a reagent blank.

EXAMPLE 16

Influence of Known Protease Inhibitors on the Activity of rFIXa UsingDifferent pHs, Alcohols and Alcohol Concentrations

Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 7.4 or 8.5

82 mmol/l NaCl

4.1 mmol/l CaCl₂

substrate MeSO₂ -D-CHG-Gly-Arg-pNA (1.02 mmol/l)

0.028-0.57 μmol/l rFIXa (human)

4.1% ethanol or 33% ethylene glycol

inhibitor at stepped concentrations (4.1-163 μmol/l)

Known synthetic inhibitors of the 3-amidinophenylalanine type were usedas inhibitors (Sturzebecher et al, J. Enzyme. Inhibition 9 (1995) 87 andWO 92/08709) (1991)). The inhibitory effect was examined at a low and ata high alcohol concentration as an example.

                  TABLE 8a                                                        ______________________________________                                        Inhibition of the activity of factor IXa (in %) by the                          inhibitor Nα-(2,4,6-triisopropylbenzenesulfonyl)-3-                     amidino-(D,L)-phenylalanine-isonipecotic acid [TIPPS-(3-                      Am)Phe-iNip-OH]                                                                         Inhibitor concentration [μmol/l]                               Conditions  4.1     8.2    16.3  40.8 81.6  163                               ______________________________________                                        pH 7.4; 4.1% ethanol                                                                      89.5    87.6   76.2  55.8 36.3  23.8                                0.57 μmol/l rFIXa                                                          pH 8.5; 4.1% ethanol 90.6 85.2 74.8 55.2 35.3 20.8                            0.28 μmol/l rFIXa                                                          pH 7.4; 33% ethylene 86.9 79.4 61.7 38.9 21.2 15.0                            glycol                                                                        0.057 μmol/l rFIXa                                                         pH 8.5; 33% ethylene 85.6 72.8 58.0 33.6 20.7 15.8                            glycol                                                                        0.028 μmol/l rFIXa                                                       ______________________________________                                    

                  TABLE 8b                                                        ______________________________________                                        Inhibition of the activity of factor IXa (in %) by the                          inhibitor Nα-(2-naphthylsulfonyl)-3-amidino-(D,L)-                      phenylalanine-1,2,3,4-tetrahydroisoquinoline-3-                               carboxylic acid [βNAPS-(3-Am)Phe-TIC-OH]                                           Inhibitor concentration [μmol/l]                               Conditions  4.1     8.2     16.3 40.8  81.6 163                               ______________________________________                                        pH 7.4; 4.1% ethanol                                                                      97.2    97.2    88.2 75.4  57.0 25.7                                0.57 μmol/l rFIXa                                                          pH 8.5; 4.1% ethanol 100   96.2 86.2 77.5 63.8 48.8                           0.28 μmol/l rFIXa                                                          pH 7.4; 33% ethylene 100   100   85.4 62.6 41.0 28.9                          glycol                                                                        0.057 μmol/l rFIXa                                                         pH 8.5; 33% ethylene 100   97.9 88.4 78.4 66.0 45.4                           glycol                                                                        0.028 μmol/l rFIXa                                                       ______________________________________                                    

Result

The inhibition of rFIXa by a synthetic inhibitor can be examined atvarious pHs (pH 7-9) and at various alcohol contents (preferably ethanolor ethylene glycol). For a 50% inhibition of rFIXa IC₅₀ values between20 and 50 μmol/l were found for the inhibitor TIPPS-(3-Am)Phe-iNip-OHand IC₅₀ values between 60 and 150 μmol/l were found for the inhibitorβNAPS-(3-Am)Phe-TIC-OH. The inhibitory effect can be detected mostsensitively at a high ethylene glycol concentration and pH 8.5. Thisapproach is suitable as a screening method for searching for FIXainhibitors since extremely low amounts of rFIXa are required and thehigh alcohol concentrations facilitate the dissolution of the substancesto be tested.

EXAMPLE 17

Determination of the Dissociation Constant K_(i) According to the Methodof DIXON

Test Mixture (Concentration in the Test)

42 mmol/l Tris-HCl, pH 8.5

82 mmol/l NaCl

4.1 mmol/l CaCl₂

33% ethylene glycol

inhibitor in stepped concentrations (0, 20.4 and 40.8 μmol/l)

MeSO₂ -D-HHT-Gly-Arg-pNA (1.02, 0.51 and 0.25 mmol/l)

0.14 μmol/l rFIXa (human)

or

MeSO₂ -D-HHT-Gly-Arg-pNA (1.02, 0.51 and 0.25 mmol/l)

0.07 μmol/l native FIXa (human)

or

MeSO₂ -D-CHG-Gly-Arg-pNA (1.02, 0.51 and 0.25 mmol/l)

0.14 μmol/l rFIXa (human)

TIPPS-(3-Am)Phe-iNip-OH was used as the inhibitor and the inhibitoryeffect was determined as an example for two substrates under optimalconditions (pH 8.5, 33% ethylene glycol).

                  TABLE 9a                                                        ______________________________________                                        Substrate: MeSO.sub.2 -D-HHT-Gly-Arg-pNA, 0.14 μmol/l rFIXa                               Inhibitor concentration                                          [μmol/l] 1/V [min/mA]                                                    Substrate concentration                                                                      0          20.4   40.8                                         ______________________________________                                        1.02 μmol/l  9.5       16.6   31.2                                           0.51 μmol/l 20.9 43.5 90.9                                                 0.25 μmol/l 33.2 69.4 137                                                ______________________________________                                    

                  TABLE 9a                                                        ______________________________________                                        Substrate: MeSO.sub.2 -D-HHT-Gly-Arg-pNA, 0.07 μmol/l native FIXa                         Inhibitor concentration                                                        [μmol/l] 1/V [min/mA]                                      Substrate concentration                                                                      0         20.4    40.8                                         ______________________________________                                        1.02 μmol/l 16.4      29.5    53.2                                           0.51 μmol/l 38.6 77.5 154                                                  0.25 μmol/l 59.5 129   255                                               ______________________________________                                    

                  TABLE 9c                                                        ______________________________________                                        Substrate: MeSO.sub.2 -D-CHG-Gly-Arg-pNA, 0.057 μmol/l rFIXa                              Inhibitor concentration                                          [μmol/l] 1/V [min/mA]                                                    Substrate concentration                                                                      0         20.4    40.8                                         ______________________________________                                        1.02 μmol/l 15.5      23.8    44.6                                           0.51 μmol/l 28.7 55.6 109                                                  0.25 μmol/l 43.1 78.1 153                                                ______________________________________                                    

Result

The determination of the dissociation constant K_(i) according to DIXONfor the inhibition of recombinant or native human FIXa byTIPPS-(3-Am)Phe-iNip-OH was carried out at pH 8.5 and at an optimalethylene glycol concentration (33%). Comparable K_(i) values (12 and 9μmol/l respectively) were determined graphically for the inhibition ofrecombinant and native FIXa using the values summarized in tables 9a and9b. Comparable Ki values (12 and 16 μmol/l respectively) also resultedwhen using the substrates MeSO₂ -D-HHT-Gly-Arg-pNA and MeSO₂-D-CHG-Gly-Arg-pNA for the inhibition of rFIXa. The selected mixturewith a high ethylene glycol concentration is especially suitable fordetermining inhibition constants since low rFIXa concentrations arerequired. High alcohol concentrations in addition facilitate thedissolution of the substances to be tested.

List of references

Bang, N. U., et al, EP 0 191 606

Bergmeyer, H. U. (ed.): Methods of Enzymatic Analysis, Vol. V, chapter3, 3rd ed., Academic Press, New York (1983)

Bharadwaj, D., et al., J. Biol. Chem. 270, 6537-6542 (1995)

Blow, D. M., Acc. Chem. Res. 9, 145-152 (1976)

Brinkmann, U., et al., Gene 85, 109114 (1989)

Davie, E. W., et al., Biochem. 30, 10363-10379 (1991)

Esmon, C. T., Prothrombin activation, doctoral dissertation, WashingtonUniversity, St. Louis, Mo. (1973)

European Patent Application No. 96 109 288.9

European Patent Application No. 96 110 959.2

European Patent EP-B 0 034 122

Fujikawa, K., et al., Biochem. 11, 4892-4898 (1972)

Furie, B.; Furie, B. C., Cell 53, 505-518 (1988)

Grodberg, J.; Dunn, J. J., J. Bacteriol. 170, 1245-1253 (1988)

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Hertzberg, M. S., et al., J. Biol. Chem. 267, 14759-14766 (1992)

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Laemmli, U. K., Nature 227, 680-685 (1970) Lin, S.-W., et al., J. Biol.Chem. 265, 144-150 (1990)

McGraw, R. A., et al., Proc. Natl. Acad. Sci. USA 82, 2847-2851 (1985)

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Pedersen, A. H., et al., Biochem. 28, 9391-9336 (1989)

Polgar, L., : Structure and function of serine proteases. In: Mechanismsof protein action. Boca Raton, Fla., CRC Press, chapter 3 (1989)

Rezaie, A. R., et al., J. Biol. Chem. 268, 8176-8180 (1993)

Rezaie, A. R.; Esmon, C. T., J. Biol. Chem. 269; 21495-21499 (1994)

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Sturzebecher, et al., WO 92/08709, 1991

Van Dam-Mieras, M. C. E., et al.: Blood coagulation factors II, V, VII,VIII, IX, X and XI: Determination with synthetic substrates. In:Bergmeyer, H. U. (ed.): Methods of Enzymatic Analysis, Vol. V, Enzymes3: Peptidases, Proteinases and Their Inhibitors, page 365-394, 3rd ed.,Academic Press, New York (1983)

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 4                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 37                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer         - - <400> SEQUENCE: 1                                                         - - aaaaaaccat ggttgttggt ggagaagatg ccaaacc      - #                       - #      37                                                                      - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 42                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer         - - <400> SEQUENCE: 2                                                         - - aaaaaaaagc ttcattaagt gagctttgtt ttttccttaa tc    - #                      - #  42                                                                      - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 39                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 3                                                         - - aaaaaaccat ggatgtaaca tgtaacatta agaatggca      - #                      - #    39                                                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:primer          - - <400> SEQUENCE: 4                                                         - - gggttcgtcc agttccagaa gggc          - #                  - #                    24                                                                    __________________________________________________________________________

I claim:
 1. A method for determining the presence of factor IXa activityin a sample solution, which comprises:(a) adding to the sample solutiona known amount of a measurable factor IXa substrate and an alcohol thatis homogeneously miscible with the sample solution; and (b) determiningif the factor IXa substrate has been cleaved, the cleavage of the factorIXa substrate indicating the presence of factor IXa activity in thesample solution.
 2. The method according to claim 1, wherein the factorIXa substrate is a tripeptide having a hydrophobic D-amino acid at theN-terminus.
 3. The method according to claim 2, wherein the factor IXasubstrate that is added is a cyclohexyl-substituted-hydrophobic D-aminoacid.
 4. The method according to claim 1, wherein the alcohol isselected from the group consisting of methanol, ethanol, n-propanol,i-propanol, t-butanol, glycerol, and ethylene glycol.
 5. The methodaccording to claim 4, wherein the alcohol is selected from the groupconsisting of methanol, ethanol, and ethylene glycol.
 6. The methodaccording to claim 5, wherein the alcohol is ethylene glycol.
 7. Themethod according to claim 5, wherein the methanol, ethanol, or ethyleneglycol is added in the range of 15% to 40% by volume of the samplesolution.
 8. A method for determining the amount of factor IXa activityin a sample solution, which comprises:(a) adding to the sample solutiona known amount of a measurable factor IXa substrate and an alcohol thatis homogeneously miscible with the sample solution; (b) determining theamount of the factor IXa substrate that has been cleaved; and (c)correlating the amount of the factor IXa substrate that has been cleavedwith a known activity of factor IXa, thereby determining the amount offactor IXa activity in the sample solution.
 9. The method according toclaim 8, wherein the factor IXa substrate is a tripeptide having ahydrophobic D-amino acid at the N-terminus.
 10. The method according toclaim 9, wherein the factor IXa substrate that is added is acyclohexyl-substituted-hydrophobic D-amino acid.
 11. The methodaccording to claim 8, wherein the alcohol is selected from the groupconsisting of methanol, ethanol, n-propanol, i-propanol, t-butanol,glycerol, and ethylene glycol.
 12. The method according to claim 11,wherein the alcohol is selected from the group consisting of methanol,ethanol, and ethylene glycol.
 13. The method according to claim 12,wherein the alcohol is ethylene glycol.
 14. The method according toclaim 12, wherein the methanol, ethanol, or ethylene glycol is added inthe range of 15% to 40% by volume of the sample solution.
 15. A methodfor increasing the factor IXa activity of a substance having factor IXaactivity, which comprises:(a) adding a homogeneously miscible alcohol toa sample solution in an amount effective to increase factor IXaactivity; and (b) reacting the substance having factor IXa activity witha factor IXa substrate in the sample solution containing thehomogeneously miscible alcohol; wherein the alcohol is selected from thegroup consisting of methanol, ethanol, n-propanol, i-propanol,t-butanol, glycerol, and ethylene glycol.
 16. The method according toclaim 15, wherein the factor IXa substrate is a tripeptide having ahydrophobic D-amino acid at the N-terminus.
 17. The method according toclaim 16, wherein the factor IXa substrate is acyclohexyl-substituted-hydrophobic D-amino acid.
 18. The methodaccording to claim 15, wherein the homogeneously miscible alcohol isselected from the group consisting of methanol, ethanol, and ethyleneglycol.
 19. The method according to claim 18, wherein the alcohol isethylene glycol.
 20. The method according to claim 18, wherein themethanol, ethanol, or ethylene glycol is in the range of 15% to 40% byvolume of the sample solution.
 21. A method for determining the abilityof a test substance to modulate factor IXa activity in a solution, whichcomprises:(a) providing a solution containing a substance having factorIXa activity and a test substance; (b) adding a factor IXa substrate tothe solution in the presence of an alcohol that is homogeneouslymiscible with the solution; (c) measuring the factor IXa activity in thesolution of step (b); (d) comparing the factor IXa activity in thesolution comprising the test substance with the factor IXa activity ofanother solution that is identical except for the absence of the testsubstance, the difference in activity representing the amount of factorIXa modulating activity attributable to the test substance.
 22. Themethod according to claim 21, wherein the factor IXa substrate is atripeptide having a hydrophobic D-amino acid at the N-terminus.
 23. Themethod according to claim 22, wherein the factor IXa substrate is acyclohexyl-substituted-hydrophobic D-amino acid.
 24. The methodaccording to claim 21, wherein the alcohol is selected from the groupconsisting of methanol, ethanol, n-propanol, i-propanol, t-butanol,glycerol, and ethylene glycol.
 25. The method according to claim 24,wherein the alcohol is selected from the group consisting of methanol,ethanol, and ethylene glycol.
 26. The method according to claim 25,wherein the alcohol is ethylene glycol.
 27. The method according toclaim 25, wherein the methanol, ethanol, or ethylene glycol is added inthe range of 15% to 40% by volume of the solution.
 28. A reagent whichcomprises a chromogenic factor IXa substrate that is cleavable by factorIXa activity, an alcohol that is homogeneously miscible with water, anda buffer wherein the reagent has a pH in the range of pH 7 to
 10. 29.The reagent according to claim 28, wherein the factor IXa substrate is atripeptide having a hydrophobic D-amino acid at the N-terminus.
 30. Thereagent according to claim 28, wherein the factor IXa substrate is acyclohexyl-substituted-hydrophobic D-amino acid.
 31. The reagentaccording to claim 28, wherein the alcohol is selected from the groupconsisting of methanol, ethanol, n-propanol, i-propanol, t-butanol,glycerol, and ethylene glycol.
 32. The reagent according to claim 31,wherein the alcohol is selected from the group consisting of methanol,ethanol, and ethylene glycol.
 33. The reagent according to claim 32,wherein the alcohol is ethylene glycol.